3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS

Article abstract of DOI:10.1021/jm201517a

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.

Full text of DOI:10.1021/jm201517a

Article
pubs.acs.org/jmc
3-Aminopiperidine-Based Peptide Analogues as the First Selective
Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS
Kristina Berggren,†,§ Reine Vindebro,‡ Claes Bergstrom,† Christian Spoerry,‡ Helena Persson,‡
̈
Tomas Fex,§ Jan Kihlberg,§ Ulrich von Pawel-Rammingen,‡ and Kristina Luthman*,†
†Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Goteborg, Sweden
̈
‡Department of Molecular Biology and Umea Centre for Microbial Research, Umea University, SE-901 87 Umea, Sweden
̊
̊
̊
§AstraZeneca R&D Molndal, SE-431 83 Molndal, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: A series of eight peptides corresponding to the amino acid
sequence of the hinge region of IgG and 17 newly synthesized peptide analogues
containing a piperidine moiety as a replacement of a glycine residue were tested as
potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus
pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but
several piperidine-based analogues were identified as inhibitors. Two different
analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage
products and a surface plasmon resonance spectroscopy based assay to quantify
the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all
compounds were screened against two other related cysteine proteases (SpeB and
papain). The selectivity results show that larger analogues that are active inhibitors
of IdeS are even more potent as inhibitors of papain, whereas smaller analogues
that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds
were identified that exhibit high selectivity against IdeS and will be used for further studies.
heavy chain. The SpeB cleavage site is identical to IdeS cleavage
at a defined site between glycine residues 236 and 237, creating
one F(ab′)2 fragment and two identical 1/2Fc fragments.6,11,12
Papain cleavage occurs at the peptide bond between histidine in
position 224 and threonine in position 225 of the hinge region
of IgG, thereby generating two Fab fragments and one Fc
fragment.13 However, the proteases have distinguished
substrate recognition properties: SpeB and papain exhibit a
broad proteolytic activity and degrade or activate a wide variety
of substrates.1,14 IdeS, on the other side, is highly specific and
recognizes only IgG as substrate.6,12,15 Furthermore, IdeS, in
contrast to papain and other prokaryotic cysteine proteases,
including SpeB and the staphylococcal cysteine protease
StpA,16 is not inhibited by the classical cysteine protease
inhibitor E64.6,12 This interesting property is explained by an
unusually narrow active site cleft that does not offer enough
space to accommodate the P3 residue of E64 and thus points to
distinct substrate recognition properties.7
INTRODUCTION
■
Proteolytic activities play a decisive role in all aspects of cellular
life, including cell division processes, metabolism and
catabolism, protein translocation, immune defense mechanisms,
and more.1 The human bacterial pathogen Streptococcus
pyogenes (or group A streptococcus) is the causative agent of
a great variety of infections, ranging from mucocutaneous
infections of the throat and skin to life threatening conditions
including necrotizing fasciitis and streptococcal toxic shock
syndrome.2 More than 600 million people are estimated to
suffer from streptococcal pharyngitis alone, and mucocutaneous
infections cause substantial morbidity and considerable
economic loss to society.3 Postinfectious sequelae include
serious inflammatory diseases such as acute rheumatic fever
(ARF), rheumatic heart disease (RHD), and poststreptococcal
glomerulonephritis (PSGN). Inflammatory autoimmune dis-
eases such as guttate psoriasis have also been associated with
streptococcal infections4 although the underlying molecular
mechanisms still remain to be solved.
S. pyogenes employs two papain-like cysteine proteases to
adapt to the dynamic environment in its human host and to
evade the human immune response: the classical streptococcal
cysteine protease SpeB and the immunoglobulin G (IgG)
degrading protease, IdeS.5,6 Both enzymes adopt a canonical
papain-like structural fold and show, despite the lack of
sequence similarity, large structural similarities.7−10 Besides
IdeS, also SpeB and papain have the ability to cleave the IgG
Given the essential role of IdeS in the evasion of IgG
mediated immune responses, there is a high medical interest to
identify specific inhibitors for prokaryotic cysteine proteases.
Furthermore, IdeS is currently evaluated as a therapeutic agent
to treat conditions in which antibodies reacting against human
antigens misdirect the human immune response toward the
Received: June 27, 2011
Published: February 27, 2012
© 2012 American Chemical Society
2549
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
body’s own cells. The efficient removal of pathogenic IgG is an
important clinical challenge, and several animal models have
provided the proof of principle for the use of IdeS as a
therapeutic agent.17−19 However, an IdeS specific inhibitor
would also allow the external control of proteolytic activity in
these applications, which might prove to be a valuable tool in
treatment.
extension both N- and C-terminally to the desired lengths of
the peptide analogues. The piperidine ring rigidifies the
structure of the analogues compared to the original peptide
backbone, yet the structure is flexible as the six-membered ring
can adopt different chair conformations. A new stereogenic
center is introduced at different positions in the two fragments
(Figure 1), and the environment around the scissile amide
bond is changed by the different polarity profiles of the
analogues. The hydrogen bond acceptor/donor ability is also
altered.
Peptides and synthesized peptide analogues were screened
for their ability to inhibit IdeS, SpeB, and papain. Interestingly,
several of the synthesized peptide analogues affected the
activity of the enzymes, however, different inhibitory profiles
were observed for the three cysteine proteases.
However, because of the structural similarity of papain-like
proteases, it is not a simple task to identify inhibitors that
efficiently block prokaryotic proteases without affecting several
essential protease functions in the human host. Compounds
reported to inhibit IdeS, including alkylating agents,6 Z-LVG
6
CHN2 and TPCK/TLCK,15 are also efficient inhibitors of
other cysteine proteases and do not exhibit any selectivity
toward IdeS. Recently, we showed that TPCK/TLCK
analogues containing aldehyde-based warheads act as reversible
inhibitors of IdeS, however their selectivity was not studied.20
The rationale for the approach in the present study was to
identify specific inhibitors for IdeS based on the fact that a
noncovalent inhibitor lacking an electrophilic warhead would
have to depend on other specific interactions with the enzyme,
which therefore should increase the selectivity and thus harbor
the potential to be specific.
IdeS does only hydrolyze IgG and neither synthetic or
natural peptides containing the P4−P1 subsites of the IgG hinge
region, nor peptides with sequences covering the IdeS cleavage
site are cleaved by the protease.12 Because such peptide-based
substrates are not hydrolyzed by IdeS, they have in the present
study instead been investigated for their putative inhibitory
capacity on the streptococcal cysteine proteases IdeS and SpeB
and also on papain. The tested peptides were of different
length, from four up to eight amino acids, covering the P4−P′4
residues of IgG. In addition, a series of di-, tri-, and tetrapeptide
analogues based on the amino acid sequence of IgG
surrounding the IdeS cleavage site have been synthesized and
were tested for potential inhibitory activity. In the analogues,
one of the two glycine residues at the cleavage site, Gly236 or
Gly237, was replaced by a piperidine moiety, thus forming either
pip236G- or Gpip237-fragments (Figure 1).
RESULTS AND DISCUSSION
■
Synthesis of Peptide Analogues. Short and efficient
synthetic routes to the peptide analogues discussed in this study
have been developed. The synthesis starts by formation of the
new stereogenic center via a reductive amination with N-
protected 3-piperidone and either glycine or enantiopure L-
proline. The stereoisomers were separated and further modified
or used in peptide coupling reactions with enantiopure L-amino
acids to form the desired peptide analogues. Separation of the
stereoisomers made it possible to keep track of the absolute
configuration of the created stereogenic center throughout the
synthesis. Further, no racemization or epimerization were
observed in any of the amino acid coupling reactions.
Reductive amination of Boc-protected 3-piperidone with
ethyl glycinate and NaBH(OAc)3 gave the racemate 1 in
moderate yield (62%) (Scheme 1). The racemate was separated
into the pure enantiomers using preparative HPLC.
To determine the absolute configuration of the two
enantiomers, reference compounds were synthesized via
reductive amination of ethyl glyoxylate with (R)- and (S)-3-
amino-1-Boc-piperidine to afford (R)- and (S)-1, respectively,
with known configuration at the stereogenic center (Scheme
1). Comparison of the retention times from analytical HPLC
allowed an unequivocal assignment of the configuration of the
enantiomers isolated via preparative HPLC. Unfortunately, the
synthesized reference compounds showed too low ee (90% and
86%, respectively) to be used as starting material for the
synthesis of the analogues. However, 2 g of (rac)-1 was
separated into (R)- and (S)-1 in 11 injections in sufficient
amount for the synthetic route to follow.
The piperidine moiety can be inserted through a short and
efficient synthetic route, and the strategy used allows further
As shown in Scheme 2, aminolysis of the isolated (R)- and
(S)-1 with ammonia in methanol afforded the amides (R)- and
(S)-2, respectively, in high yields (92% and 90%) without the
need for purification. The 13C NMR spectra showed a
considerable broadening of the signals assigned to the carbon
atoms adjacent to the nitrogen in the piperidine ring, indicating
that the Boc-group gives rise to rotamers. Removal of the Boc
protecting group by TFA gave the GlyGly-NH2 analogues (R)-
1
and (S)-pipG (3) in quantitative yields. The H NMR spectra
showed high purity for both compounds. Furthermore, the
1
coupling pattern in the H NMR spectra implies that the
piperidine ring adopts one single chair conformation with the
glycine amide residue in an equatorial position. The suggested
ring conformation is shown in the Supporting Information.
Routes to the analogues of the tripeptide LeuGlyGly-NH2,
(R)- and (S)-LpipG (7), and the tetrapeptide LeuLeuGlyGly-
NH2, (R)- and (S)-LLpipG (10), are shown in Scheme 2. The
Figure 1. In the synthesized analogues, a piperidine moiety replaces
one of the two glycine residues at the IdeS cleavage site. Thereby, a
new stereogenic center is introduced at different positions in the two
fragments (marked with an asterisk).
2550
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (a) ethyl glycinate, NaBH(OAc)3, AcOH, molecular sieves, dichloromethane, room temp, 20 h (62%); (b) preparative
HPLC; (c) ethyl glyoxylate, NaBH(OAc)3, AcOH, dichloromethane, room temp, 4 h (56% and 62%).
a
Scheme 2
a
Reagents and conditions: (a) NH3 in methanol, room temp, 72 h (92 and 90%); (b) TFA, room temp, 12 h (quant yields); (c) Cbz-OSu, Et3N,
dichloromethane, 0 °C → room temp, 17 h (82% and 84%); (d) (i) TFA, room temp, 16 h, (ii) Boc-L-Leu × H2O, EDC, HOBt, dichloromethane,
molecular sieves, Et3N, room temp, 15 h (91% and 79% over two steps); (e) NH3 in methanol, room temp, 72 h (65% and 73%); (f) (i) H2, Pd/C,
ethanol, room temp, 72 h, (ii) TFA, room temp, 4 h (84% and 75% over two steps); (g) (i) TFA, room temp, 16 h, (ii) Boc-L-Leu × H2O, EDC,
HOBt, dichloromethane, molecular sieves, Et3N, room temp, 15 h (88 and 91%, over two steps); (h) NH3 in methanol, room temp, 168 h (63% and
86%); (i) (i) H2, Pd/C, ethanol, 3 h, (ii) TFA, room temp, 14 h (90% and 92% over two steps).
isolated enantiomers (R)- and (S)-1 were protected using Cbz-
succinimide (Cbz-OSu) to afford (R)- and (S)-4, respectively,
in high yields (82% and 84%). The 13C NMR spectra recorded
at ambient temperature showed rotamers from both the Boc-
and Cbz-groups, however the signals coalesced in spectra
recorded at 50 °C. The enantiomers (R)- and (S)-4 were Boc-
deprotected followed by coupling with Boc-L-leucine to afford
high yields of (R)- and (S)-5, respectively (91% and 79%).
Aminolysis afforded amides (R)- and (S)-6, which were
deprotected to the end products (R)- and (S)-LpipG (7),
respectively (yields: 84% and 75% over two steps). Boc-
Deprotection of (R)- and (S)-5 and coupling to Boc-L-leucine
gave (R)- and (S)-8, respectively, in high yields (88% and
91%). A subsequent aminolysis afforded (R)- and (S)-9. The
reaction times were rather long and purification was needed
which resulted in lower yields (63% and 86%). Finally, the Cbz-
and Boc-protecting groups were removed by catalytic hydro-
genation (Pd/C) and TFA hydrolysis, respectively, to afford
the end products (R)- and (S)-LLpipG (10) in high yields
(90% and 92%). The total yield for the synthesis of (R)- and
(S)-LLpipG (10) over eight steps from (R)- and (S)-1,
respectively, was 37% and 48%.
N-Ethylated byproducts were formed during the Cbz-
deprotection to (R)- and (S)-LpipG (7) as well as (R)- and
(S)-LLpipG (10) using catalytic hydrogenation (Pd/C 10%).
Isolation of the byproducts after prolonged reaction time and
subsequent TFA hydrolysis gave (R)- and (S)-LLpipEtG (11)
(Scheme 3). Already after three hours, byproduct formation
was observed. To verify the structure of the byproduct, Cbz-
deprotected (R)-9 was converted to the corresponding
ethylated product in 76% yield via reductive amination using
acetaldehyde and NaBH(OAc)3 (reaction not shown). The
NMR spectra of the synthesized compound and that of (R)-
LLpipEtG (11) were identical. It has been proposed that a Pd-
catalyzed oxidation of ethanol to acetaldehyde can occur during
the hydrogenation reaction.21,22 The acetaldehyde forms an
2551
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
a
78%). A small portion of (R)- and (S)-15 were deprotected to
provide the GlyGlyPro-NH2 analogues (R)- and (S)-pipGP
(16). Interestingly, the NMR spectra of the two diastereomers
were highly similar. Furthermore, the coupling pattern in the
1H NMR spectra resembled the pattern seen for (R)- and (S)-
pipG (3), implying that also here the monosubstituted
piperidine ring adopts a defined chair conformation. To
continue, removal of the Boc-protecting groups in (R)- and
(S)-15 and subsequent couplings with Boc-L-leucine mediated
by HOBt and EDC gave the epimers (R)- and (S)-17 in
moderate to high yields (64% and 90%, respectively). The
lower yield is due to the need of two purification steps. Also
here, the NMR spectra recorded at different temperatures
Scheme 3
a
Reagents and conditions: (a) (i) H2, Pd/C, ethanol, room temp, 144
h (23 and 13%), (ii) TFA in dichloromethane, room temp, 16 h (99%
and 98%).
imine with the Cbz-deprotected secondary amine, and a
subsequent reduction provides the ethylated byproduct.
The unsaturated enamine byproduct 12 (Scheme 4) was
collected from a reductive amination reaction of 1-Boc-3-
1
identified rotamers. The H NMR spectrum of (R)-17 showed
five doublets assigned to the two methyl groups in the leucine
side chain, implying that the molecule adopts several different
conformations at room temperature. Finally, the two protecting
groups were removed as described above to give the end
products (R)- and (S)-LpipGP (18) in good yields (72% and
73%, respectively) both with excellent purities (100%) when
analyzed by reversed phase HPLC. After the deprotection, the
1H NMR spectrum of both (R)- and (S)-LpipGP (18) showed
two doublets assigned to the two nonequivalent methyl groups,
as expected. The total yield for the synthesis of (R)- and (S)-
LpipGP (18) over seven steps was 29% and 43%, respectively.
The corresponding tetrapeptide analogues in which the
piperidine moiety replaces Gly237 in IgG ((R)- and (S)-LGpipP
(23)) were also synthesized (Scheme 6). Reductive amination
of Boc-protected 3-piperidone with L-proline amide and
NaBH(OAc)3 afforded 19 in 68% yield and a diastereomeric
ratio of 60:40. The diastereomers were separated by preparative
HPLC to give (R)- and (S)-19, and the absolute configurations
were determined by chemical correlation.23
Boc-protected glycine was coupled to each deprotected
diastereomer using EDC and HOBt to afford (R)- and (S)-20
in 45% and 47% yield, respectively. Deprotection with TFA in
dichloromethane gave quantitative yields of (R)- and (S)-
GpipP (21) as TFA salts. A subsequent coupling to Boc-
protected L-leucine gave (R)- and (S)-22, respectively, in high
yields (93% and 78%). Deprotection with TFA in dichloro-
methane overnight afforded (R)- and (S)-LGpipP (23) in
excellent yields. The total yield for the synthesis of (R)- and
a
Scheme 4
a
Reagents and conditions: (a) NH3 in methanol, room temp, 72 h
(93%); (b) TFA/dichloromethane, room temp, 12 h (97%).
piperidone with methyl glycinate performed in a highly
concentrated reaction mixture. The opportunity to use the
unsaturated piperidine as a scaffold for the synthesis of
analogues was taken. Attempts to Cbz-protect 12 using the
conditions described for (S)-4 gave only unreacted starting
material back. Boc-Deprotection by TFA in dichloromethane
was performed, but subsequent coupling with Boc-L-leucine did
not provide satisfying results. However, 12 was amidated by
treatment with NH3 in methanol and subsequent Boc-
deprotection gave pip(db)G (13) in 90% (Scheme 4).
The synthetic route to the LeuGlyGlyPro-NH2 analogues
(R)- and (S)-LpipGP (18), in which Gly236 in IgG is mimicked
by the piperidine moiety, is shown in Scheme 5. Starting from
the Cbz-protected core compounds (R)-and (S)-4, hydrolysis
of the esters in excellent yields (95% and 99% of (R)- and (S)-
14, respectively) followed by coupling to L-proline amide
afforded (R)- and (S)-15, respectively, in high yields (82% and
a
Scheme 5
a
Reagents and conditions: (a) LiOH 1 M aq, THF/MeOH/H2O 3:1:1, 0 °C 30 min, room temp, 3 h (95 and 99%); (b) L-Pro-NH2, EDC, HOBt,
Et3N, dichloromethane, room temp, 16 h (82% and 78%); (c) (i) H2, Pd/C, ethanol, room temp, 24 h, (ii) TFA, room temp, 4 h (quant yields over
two steps); (d) (i) TFA, room temp, 16 h, (ii) Boc-L-Leu × H2O, EDC, HOBt, dichloromethane, molecular sieves, Et3N, room temp, 15 h (64% and
90% over two steps); (e) (i) H2, Pd/C, ethanol, room temp, 48 and 24 h, (ii) TFA, room temp, 4 h (72% and 73% over two steps).
2552
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
a
Scheme 6
a
Reagents and conditions: (a) (i) L-Pro-NH2, NaBH(OAc)3, HOAc, dichloromethane, room temp, 20 h (68%), (ii) preparative HPLC; (b) (i)
TFA/dichloromethane, room temp, (ii) Boc-Gly, EDC, HOBt, dichloromethane, 0 °C → room temp, 15 h, 45 and 47% over two steps); (c) TFA/
dichloromethane, room temp, 20 h (quant yields); (d) (i) TFA/dichloromethane, room temp, 20 h (quant yields), (ii) Boc-L-Leu, EDC, HOBt,
Et3N, dichloromethane, 0 °C → room temp, 15 h (93% and 78%); (e) TFA/dichloromethane, room temp, 12 h (quant yield and 98%).
(S)-LGpipP (23) in five steps from (R)- and (S)-19 was 42%
and 36%, respectively.
IgG-Based Peptides As Potential Inhibitors of IdeS.
Eight different peptides based on the amino acid sequence of
the IgG hinge region (Figure 2) were tested for putative
inhibitory activity toward IdeS.24
Figure 2. Peptides tested for inhibitory activity.
The inhibitory activity was screened in a gel-electrophoresis
(SDS-PAGE) based assay to detect IgG cleavage products.25
None of the peptides was found to be active as inhibitors of
IdeS (data not shown). Thus, although the sequences of the
tested peptides are identical to the IgG sequence around the
IdeS cleavage site, these short peptides did apparently not bind
strongly enough to the enzyme to affect enzymatic activity.
Inhibitory Capacity of Peptide Analogues toward
IdeS. In total 17 piperidine-containing analogues of the peptide
Figure 3. (A) Representative nonreducing SDS-PAGE analysis of IgG
sequences GG, LLGG, and LGGP, all based on the IdeS
cleavage site in human IgG1, were tested for their inhibitory
activity toward IdeS. Inhibitory effects were screened by SDS-
PAGE and quantified by surface plasmon resonance spectros-
copy (Figure 3).15
and IgG hydrolyzed by IdeS in the presence ((S)-pipG ((S)-3)) or
absence (DMSO) of peptide analogues. scIgG indicates single-chain
cleaved IgG; F(ab′)2 indicates double-chain cleaved IgG. (B)
Representative surface plasmon resonance spectroscopy curves of
uncleaved IgG (control) and cleaved IgG in presence of peptide
analogues (S)-pipG ((S)-3) and (R)-GpipP ((R)-21). Differences in
response units are indicated by arrows. (S)-pipG ((S)-3) has greater
inhibitory activity than (R)-GpipP ((R)-21).
Whereas the corresponding peptides were inactive, the
peptide analogues showed a significant inhibitory activity
(Figure 4). The most potent inhibitors of IdeS activity are
(R)-pipGP ((R)-16), (S)-LpipGP ((S)-18), (S)-pipGP ((S)-
16), (S)-pipG ((S)-3), and (R)-LpipG ((R)-7). In general,
peptide analogues in which the piperidine moiety replaces the
glycine corresponding to G236 in IgG (pip236G) appear to be
stronger inhibitors than analogues with the piperidine moiety
replacing G237 of IgG (Gpip237), independently of C-terminal or
N-terminal amino acid extensions. For example, (R)-pipGP
((R)-16) is more active than (R)-GpipP ((R)-21) and (S)-
LpipGP ((S)-18) is more active than (S)-LGpipP ((S)-23).
The two (R)- and (S)-epimers of each analogue pair were
tested, but no clear trend how stereochemistry of the piperidine
moiety affected potency could be seen (Figure 4).
The ethylated analogues (R)- and (S)-LLpipEtG (11) have
also been tested, both showing comparable inhibitory activity
against IdeS as (S)-LLpipG ((S)-10). This implies that the
secondary amine is not involved in hydrogen bond donation
and that there is space enough for an ethyl group. Interestingly,
the structural difference between LpipGP (18) and pipG (3) is
two amino acids, but they show similar inhibitory activity. This
2553
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
extended structural similarity of papain-like proteases raised the
question whether piperidine containing peptide analogues
could have inhibitory capacity toward other IgG cleaving
proteases or in fact harbor a potential specificity toward IdeS.
Therefore, peptides and peptide analogues were also screened
for activity toward the streptococcal cysteine protease SpeB and
the family protease papain (Figure 5).
Interestingly, very different inhibitory profiles were obtained
that apparently distinguish the proteases from the profile
obtained for IdeS and also from each other (Figures 4 and 5).
In general, the inhibitory effect of all the tested compounds was
much less pronounced on SpeB compared to inhibition of IdeS.
Hardly any of the pip236G-fragment containing analogues had
any inhibitory effect on SpeB activity (Figure 5A). Notable is
however that an inhibitory capacity was observed for the LLGG
peptide. In contrast to the effect observed on IdeS activity
(Figure 4), the degree of inhibition was not improved when
testing LLpipG (10) peptide analogues in which Gly236 was
replaced with a piperidine moiety. However, a clearly improved
degree of inhibition could be observed when testing the
ethylated analogues (R)- and (S)-LLpipEtG (11). In addition,
(R)- and (S)-LGpipP (23) containing a similar tertiary amine
fragment as the ethylated analogues also inhibited SpeB activity,
which is in contrast to the effect observed for IdeS. Thus, the
absence of the possibility of hydrogen bond donation and the
rigid structure of neighboring piperidine and proline rings
appear to increase the interaction between the LGpipP (23)
analogues and SpeB. Consequently, LpipGP (18) analogues do
not affect SpeB activity.
Figure 4. IdeS activity in presence of peptides and peptide analogues
using IgG1 as substrate. IdeS activity is expressed relative to a standard
curve obtained in absence of inhibitors. (R) and (S) indicate the
different stereoisomers of each analogue; “pip” represents the
piperidine moiety replacing glycine residues. The enzyme activity
was determined for IdeS at a final concentration of 0.8 μM in the
presence of 4.8 × 103 molar excess of peptides/peptide analogues.
indicates that the extra amino acids are not contributing to
more favorable interactions to accomplish more efficient
binding or are too flexible to gain favorable binding energies.
Thus, when evaluating ligand efficiency (LE), where binding
energy per atom is considered, (S)-pipG ((S)-3) is the most
favorable peptide analogue in the series.26
The pKa values for (R)- and (S)-pipG (3) and (R)- and (S)-
LpipGP (18) have been determined (Table 1). Interestingly,
the two pKa values differ considerably. For (R)- and (S)-pipG
(3), the acyclic amine bears the electron withdrawing acetamide
group, thus the piperidine nitrogen would be expected to be
protonated first. For (R)- and (S)-LpipGP (18), the primary
amine would be expected to have the pKa of an amino acid
amide, i.e., between 8 and 9, which is what is found. The acyclic
secondary amines will not be protonated at physiological pH,
and the pip moiety is therefore able to act as a glycine isostere.
Specificity of Peptide Analogues. IdeS exhibits pro-
nounced substrate specificity in comparison to other IgG
cleaving cysteine proteases of the papain superfamily.12 Still, the
The specificity study was extended further to include the
cysteine protease papain. Like SpeB also, papain exhibits a
broad proteolytic activity and degrades a wide variety of
substrates, including IgG. The analysis of putative inhibitory
effects of peptides and piperidine-containing analogues on
papain revealed that the enzyme was clearly very sensitive
toward most compounds tested (Figure 5B). In analogy to
SpeB, the positioning of the piperidine in the peptide sequence
favors a replacement of the glycine corresponding to G237
,
resulting in the most efficient papain inhibitors in the series,
with (R)-LGpipP ((R)-23) being the most active analogue.
In summary, the most active inhibitors of IdeS, (R)-pipGP
((R)-16) and (S)-LpipGP ((S)-18), are even more active
toward papain but show no inhibitory activity toward SpeB.
a
Table 1. pKa Values for the pipG and LpipGP Analogues
measured
predicted
compd
pKa1
pKa2
pKa1
pKa2
(R)-pipG
4.41
4.35
5.13
5.21
10.11
10.06
8.46
5.40
9.84
(S)-pipG
(R)-LpipGP
7.35
9.13
(S)-LpipGP
8.53
a
Determination of pKa values performed by a screening method based on pressure-assisted capillary electrophoresis and mass spectrometry.27
Prediction of pKa values was performed using the ACD/Laboratories software (Advanced Chemistry Development, Inc.).28
2554
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
unchanged by the presence of the inhibitors and therefore Ki
values could not be determined reliably, but the values exceed
those observed for IdeS at least 16−267-fold, respectively.
Thus, the aminopiperidine containing analogues (S)-pipG
((S)-3) and (R)-LpipG ((R)-7) exhibit effective and specific
IdeS inhibitor properties and will serve as a starting point for
further development of more potent inhibitors. Initially, an
attempt to increase the rigidity of the compound will be
undertaken which hopefully will increase the potency of the
inhibitor. Synthesis of such compounds is ongoing.
CONCLUSION
■
A series of 17 piperidine-containing peptide analogues have
been efficiently synthesized. The peptide analogues were tested
in inhibition assays addressing cysteine protease activities with
focus on the streptococcal IgG degrading enzyme IdeS. Several
of the synthesized piperidine-containing peptide analogues are
active IdeS inhibitors, but the piperidine moiety does not
necessarily improve the inhibitory capacity of the analogues
toward other cysteine proteases. To the best of our knowledge,
these are the first noncovalently bound inhibitors of IdeS, also
showing selectivity toward SpeB and papain.
EXPERIMENTAL SECTION
■
Synthesis. General. Starting materials and reagents were
purchased from Sigma-Aldrich or Bachem and were used as such.
The peptides were purchased from CASLO Laboratory ApS, Lyngby,
Denmark. The purity of the peptides was >98%. Analytical TLC was
performed on silica plated aluminum sheets (grade 60 F254, Merck).
The spots were visualized by treatment with a dip solution [KMnO4
(1.5 g), K2CO3 (10 g), NaOH (10%) (1.25 mL) in water (200 mL)],
followed by heating. Flash chromatography was performed on Merck
silica gel 60 (0.040−0.063 mm). 1H and 13C NMR spectra were
recorded on a JEOL Eclipse 400 spectrometer at 400 and 100 MHz,
respectively, at ambient temperature and in CDCl3 if not otherwise
stated. 13C NMR spectra were also recorded on a Varian 500
spectrometer at 125 MHz. Chemical shifts are reported in ppm, with
the solvent residual peak as internal standard (CHCl3 δH 7.26, δC 77.0,
CH3OH δH 3.30, δC 49.0). DEPT, COSY, TOCSY, and HSQC spectra
were recorded to validate structural assignments of the NMR signals.
Separation of the stereoisomers of 1 and 19 were performed on a
preparative HPLC Shimadzu system equipped with a Chiralpak AD
250 mm × 20 mm column (Daicel Chemical CO., Tokyo, Japan) with
a mobile phase consisting of hexane/ethanol 95:5 or hexane/2-
propanol 90:10 at a flow rate of 15 or 10 mL/min, respectively.
Detection was carried out at 240 nm. Chiral HPLC analysis was
performed on a Varian system equipped with a Chiralpak AD 4.6 mm
× 250 mm column (Daicel Chemical CO., Tokyo, Japan) at a flow rate
of 1 mL/min. Detection was carried out at 240 nm. Analytical reversed
phase HPLC was performed on a Waters 2690 system (photodiode
array detector at 220 nm and flow rate 1.0 mL/min) equipped with a
250 mm × 4.6 mm Reprosil PUR C18aq column. Optical rotation was
measured with a Perkin-Elmer 341 LC polarimeter at 20 °C. Infrared
spectra were recorded on a Perkin-Elmer 16 PC FTIR spectrometer,
and only the major peaks are listed. HRMS analyses were run at
BioAnSer, Gothenburg, Sweden. The purity of all tested compounds
was determined using analytical reversed phase HPLC on a Waters
2690 system (photodiode array detection at 220 nm and a flow rate of
1.0 mL/min) equipped with a 250 mm × 4.6 mm Reprosil PUR
C18aq column. (S)- and (R)-pipG (3) were further analyzed using a
reversed phase Waters Acquity UPLC system equipped with a BEC
C18 column, connected to a MS instrument with a Waters SQD single
quadrupole detector. All compounds showed a purity >95% except
(S)-pipG ((S)-3) which was 90% pure. Measurement of the aqueous
dissociation constant, pKa values, of (R)- and (S)-LpipGP (18) and
Figure 5. Inhibitory profile of peptides and peptide analogues on the
activity of streptococcal cysteine protease SpeB (A) and family class
protease papain (B). Activity is expressed relative to enzyme activity in
absence of putative inhibitors. (R) and (S) indicate the different
stereoisomers of each analogue; “pip” represents the piperidine moiety
replacing glycine residues. The enzyme activity was determined for
papain at a final concentration of 0.9 μM and SpeB at final
concentration of 0.28 μM in the presence of 4.8 × 103 molar excess
of peptides/peptide analogues.
However, both (S)-pipG ((S)-3) and (R)-LpipG ((R)-7) show
promising selectivity profiles as they are both potent inhibitors
of IdeS but are hardly affecting papain and do not inhibit SpeB.
These compounds were investigated further to evaluate
whether they might be a suitable starting point for the
development of specific IdeS protease inhibitors.
Various amounts of inhibitor and substrate were used to
determine the inhibition constant (Ki) for these two
compounds. Because a mechanism of competitive inhibition
was postulated, initial velocities were determined in the
presence or absence of inhibitor. The binding affinity of IdeS
for IgG has been previously determined, and a high affinity was
observed with a Kd of 2.5 μM and a Km for complete enzymatic
cleavage ranging from 6.8 to 18.9 μM depending on the IgG
subtype.12
Because of the high affinity substrate binding, a considerable
excess of inhibitor was expected to be necessary to affect initial
velocities and to successfully compete for IgG. The Ki values for
(S)-pipG ((S)-3) and (R)-LpipG ((R)-7) were determined to
approximately 6.7 and 5.7 mM, respectively. Although these
concentrations are too high for in vivo evaluation, they were
surprisingly low considering the high affinity of IdeS for IgG.
Importantly, initial velocities of papain activity remained
(R)- and (S)-pipG (3) were performed at AstraZeneca, R&D Molndal,
̈
Sweden.27
2555
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
Ethyl 2-[(1-tert-Butoxycarbonylpiperidin-3-yl)amino]acetate
((rac)-1). NaBH(OAc)3 (5.3 g, 25.0 mmol) was added to a solution of
1-Boc-piperidin-3-one (4.98 g, 25.0 mmol), acetic acid (2.25 g, 37.5
mmol), glycine ethyl ester hydrochloride (3.5 g, 25.0 mmol), and
molecular sieves (4.5 g) in dichloromethane (100 mL). The reaction
mixture was stirred at room temperature for 20 h, diluted with
dichloromethane (100 mL), and quenched by the addition of aqueous
NaOH (3 M) (20 mL). The aqueous phase was extracted with
dichloromethane (2 × 100 mL). The combined organic phases were
dried (Na2SO4), filtered, and concentrated in vacuo. Purification by
flash chromatography (heptane/ethyl acetate 1:2, Rf = 0.18) gave
(rac)-1 as a transparent oil (4.4 g, 62%). Resolution of a portion of the
racemate (2.0 g) by chiral preparative HPLC gave the enantiomers of
1.
(−)-Ethyl 2-[(1-tert-Butoxycarbonyl-(R)-piperidin-3-yl)-
amino]acetate ((R)-1). Resolution of (rac)-1 (2.0 g) gave
enantiomerically pure (R)-1 (779 mg). Analytical chiral HPLC
(hexane/2-propanol 95:5) tR 9.4 min; 95% purity, ee 100%. 1H
NMR δ 4.18 (q, J = 7.1 Hz, 2H), 3.80−3.73 (m, 2H), 3.55−3.41 (AB
system, 2H), 2.86−2.51 (m, 3H), 2.20−1.80 (m, 2H), 1.73−1.64 (m,
1H), 1.47−1.21 (m, 2H), 1.44 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H). 13C
NMR δ 172.2, 154.6, 79.2, 60.6, 53.3, 48.6, 48.1, 43.9, 31.1, 28.2, 23.2,
14.0; [α]D20 −8.5 (c 1.0, CHCl3). IR (neat) v 3515, 3333, 2976, 2933,
2860, 1738, 1691, 1423, 1157 cm−1. HRMS (FT-ICR-MS) calcd for
C14H26N2O4 [M + H]+ 287.1965; found 287.1962. For NMR spectral
assignments, see the Supporting Information.
3.7, 3.7 Hz, 1H), 1.97−1.84 (m, 1H), 1.73−1.63 (m, 1H), 1.51−1.23
(m, 2H), 1.45 (s, 9H). 13C NMR δ 175.1, 154.6, 79.2, 53.7, 49.5, 48.7,
43.8, 30.9, 28.1, 22.9; [α]D −4.6 (c 1.0, CHCl3). IR (KBr) v 3398,
20
2977, 2933, 1683, 1638, 1423, 1262, 1180, 1155 cm−1. HRMS (FT-
ICR-MS) calcd for C12H23N3O3 [M + H]+ 258.1812; found 258.1809.
For NMR spectral assignment, see the Supporting Information.
(+)-2-[(1-tert-Butoxycarbonyl-(S)-piperidin-3-yl)amino]-
acetamide ((S)-2). NH3 in methanol (7 N) (4.3 mL) was added to
(S)-1 (214 mg, 0.75 mmol), and the reaction mixture was stirred at
room temperature for 72 h. The solvent and excess of reagent were
removed in vacuo. The remaining was trituated with hexane/diethyl
ether (1:1) (1 mL) to give (S)-2 as a white solid (173 mg, 90%).
Analytical chiral HPLC (hexane/ethanol 97:3) tR 76.0 min, purity
100%. 1H NMR δ 7.06 (br s, 2H), 5.62 (br s, 1H), 3.94−3.56 (m, 2H),
3.39−3.24 (AB system, 2H), 3.32−2.72 (m, 2H), 2.56 (app tt, J = 8.1,
8.1, 3.7, 3.7 Hz, 1H), 1.95−1.86 (m, 1H), 1.73−1.57 (m, 2H), 1.45 (s,
9H), 1.39−1.23 (m, 2H). 13C NMR δ 175.0, 154.5, 79.1, 53.6, 49.4,
20
48.6, 43.7, 30.8, 28.0, 22.8; [α]D +5.1 (c 1.0, CHCl3). IR (KBr) v
3398, 2977, 2933, 1683, 1638, 1423, 1262, 1180, 1155 cm−1. HRMS
(FT-ICR-MS) calcd for C12H23N3O3 [M + H]+ 258.1812; found
258.1810.
(−)-2-((R)-Piperidin-3-ylamino)acetamide × 2TFA ((R)-pipG)
((R)-3). Deprotection of (R)-2 (137 mg, 0.53 mmol) using TFA (364
mg, 3.2 mmol) was performed as described for (S)-pipG ((S)-3) to
give the TFA salt of (R)-pipG ((R)-3) as a white solid (210 mg,
quant). 1H NMR (CD3OD) δ 4.00−3.88 (AB system, 2H), 3.73 (dd, J
= 12.1, 4.0 Hz, 1H), 3.63−3.51 (app tt, J = 11.4, 11.4, 4.1, 4.1 Hz, 1H),
3.40 (app dd, J = 12.9, 3.6 Hz, 1H), 3.08 (app t, J = 11.7 Hz, 1H), 2.96
(dt, J = 12.7, 12.7, 3.2 Hz, 1H), 2.32−2.24 (m, 1H), 2.14−2.04 (m,
1H), 1.87−1.78 (m, 1H), 1.71 (app ddt, J = 12.5, 12.5, 3.6 Hz, 1H).
(+)-Ethyl 2-[(1-tert-Butoxycarbonyl-(S)-piperidin-3-yl)-
amino]acetate ((S)-1). Resolution of (rac)-1 (2.0 g) gave
enantiomerically pure (S)-1 (802 mg). Analytical chiral HPLC
(hexane/2-propanol 95:5) tR 8.7 min, 94% purity, ee 100%. 1H
NMR δ 4.18 (q, J = 7.1 Hz, 2H), 3.81−3.73 (m, 2H), 3.51−3.37 (AB
system, 2H), 2.93−2.48 (m, 3H), 2.03−1.80 (m, 3H), 1.73−1.64 (m,
1H), 1.49−1.21 (m, 10H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR δ 172.2,
20
13C NMR (CD3OD) δ 168.5, 53.0, 46.6, 45.0, 44.6, 26.2, 21.6; [α]D
−9.3 (c 0.41, CH3OH). IR (neat) v 3399, 3012, 2822, 1683, 1436,
1205, 1130 cm−1. HRMS (FT-ICR-MS) Calcd for C7H15N3O [M +
H]+ 158.1288; found 158.1287; pKa1 = 5.13, pKa2 = 8.46. For NMR
spectral assignment, see the Supporting Information.
20
154.6, 79.1, 60.5, 53.3, 48.9, 48.1, 43.9, 31.0, 28.2, 23.2, 13.9; [α]D
+8.5 (c 1.0, CHCl3). IR (neat) v 3515, 3333, 2976, 2933, 2860, 1738,
1691, 1423, 1157 cm−1. HRMS (FT-ICR-MS) calcd for C14H26N2O4
[M + H]+ 287.1965; found 287.1962.
(+)-2-((S)-Piperidin-3-ylamino)acetamide × 2TFA ((S)-pipG)
((S)-3). TFA (375 mg, 3.3 mmol) was added to (S)-2 (141 mg, 0.55
mmol) in dichloromethane (3 mL), and the reaction mixture was
stirred at room temperature for 12 h. The solvent and excess of
reagent were removed in vacuo to give the TFA-salt of (S)-pipG ((S)-
3) as a white solid (210 mg, quant). 1H NMR (CD3OD) δ 4.00−3.85
(AB system, 2H), 3.76−3.65 (m, 1H), 3.56 (app tt, J = 11.4, 11.4, 3.7,
3.7, 1H), 3.44−3.34 (m, 1H), 3.08 (app t, J = 11.7 Hz, 1H), 2.96 (app
dt, J = 12.8, 12.8, 3.3 Hz, 1H,), 2.33−2.21 (m, 1H), 2.15−2.01 (m,
1H), 1.88−1.60 (m, 2H). 13C NMR (CD3OD) δ 168.4, 53.0, 46.6,
45.0, 44.6, 26.2, 21.6; [α]D20 +6.6 (c 0.41, CH3OH). IR (neat) v 3383,
3014, 2824, 1676, 1432, 1202, 1130 cm−1. HRMS (FT-ICR-MS) calcd
for C7H15N3O [M + H]+ 158.1288; found 158.1287. The purity was
90% according to UPLC analysis. pKa1 = 5.21, pKa2 = 8.53.
(−)-Ethyl 2-[(Benzyloxycarbonyl)(1-tert-butoxycarbonyl-(R)-
piperidin-3-yl)amino]acetate ((R)-4). Compound (R)-4 was
synthesized from (R)-1 (802 mg, 2.8 mmol) and N-
(benzyloxycarbonyloxy)succinimide (698 mg, 2.8 mmol) with Et3N
(312 mg, 3.1 mmol) in dichloromethane (11 mL) as described for (S)-
4 to give the product as a transparent oil (0.98 g, 82%). Analytical
chiral HPLC (hexane/2-propanol 95:5) tR 19.8 min, 98% purity, ee
100%. 1H NMR (rotamers) δ 7.59−7.20 (m, 5H), 5.19 and 5.10 (rot,
s, 2H), 4.34−3.72 (m, 6H), 2.80−2.43 (m, 3H), 2.03−1.87 (m, 1H),
1.80−0.98 (m, 1H), 1.43−1.17 (m, 2H), 1.43 and 1.37 (rot, s, 9H),
1.26 and 1.17 (rot, t, J = 7.1 Hz, 3H). 13C NMR δ 169.7, 155.8, 155.1,
154.5, and 154.3 (rot), 136.1, 128.3, and 128.2 (rot), 127.8, 127.5,
79.4, 67.4, and 67.1 (rot), 60.9, 53.1, and 52.9 (rot), 47.0 and 46.5,
45.6 and 45.3, 43.2, 28.7, 28.1, 24.4, 13.9; [α]D20 −14.9 (c 1.0, CHCl3).
IR (neat) v 3500, 2976, 2864, 1749, 1693, 1416, 1152 cm−1. HRMS
(FT-ICR-MS) calcd for C22H32N2O6 [M + H]+ 421.2333; found
421.2333. For NMR spectral assignment, see the Supporting
Information.
Both enantiomers of 1 were also obtained separately by reductive
alkylation of 3-amino-1-tert-butyloxycarbonylpiperidine with defined
stereochemistry. These products were used as reference material.
(−)-Ethyl 2-[(1-tert-Butoxycarbonyl-(R)-piperidin-3-yl)-
amino]acetate ((R)-1). Ethyl glyoxylate in toluene (50%) (0.5 mL,
2.5 mmol) was added dropwise to a mixture of (R)-3-amino-1-tert-
butyloxycarbonylpiperidine (500 mg, 2.5 mmol) and Na2SO4 (900
mg) in dichloromethane (9 mL). The reaction mixture was stirred at
room temperature for 2 h. NaBH(OAc)3 (529 mg, 2.5 mmol) and
AcOH (225 mg, 3.74 mmol) were added, and the stirring continued
for 90 min. The mixture was diluted with dichloromethane (20 mL)
and quenched by addition of aqueous NaOH (3 M) (8 mL). The
aqueous phase was extracted with dichloromethane (1 × 20 mL). The
combined organic phases were dried (Na2SO4) and filtered and the
solvent was removed in vacuo. Purification by flash chromatography
(heptane/ethyl acetate 1:3, Rf = 0.17) gave (R)-1 as a transparent oil
(410 mg, 60%). Analytical chiral HPLC (hexane/2-propanol 95:5) tR
9.3 min; ee 90%. NMR data were in agreement with those reported for
the pure enantiomer (R)-1.
(+)-Ethyl 2-[(1-tert-Butoxycarbonyl-(S)-piperidin-3-yl)-
amino]acetate ((S)-1). Compound (S)-1 was synthesized from (S)-
3-amino-1-tert-butyloxycarbonylpiperidine (500 mg, 2.5 mmol) using
the same procedure as described for (R)-1 to afford (S)-1 as a
transparent oil (380 mg, 56%). Analytical chiral HPLC (hexane/2-
propanol 95:5) tR 8.5 min; ee 86%. NMR data were in agreement with
those reported for the pure enantiomer (S)-1.
(−)-2-[(1-tert-Butoxycarbonyl-(R)-piperidin-3-yl)amino]-
acetamide ((R)-2). Compound (R)-2 was synthesized from (R)-1
(210 mg, 0.73 mmol) and NH3 in methanol (7 N) (4.2 mL) as
described for (S)-2 to give the product as a white solid (174 mg, 92%).
Analytical HPLC (hexane/ethanol 97:3) tR 84.4 min, purity 100%. 1H
NMR δ 7.05 (br s, 2H), 5.53 (br s, 1H), 3.97−3.54 (m, 2H), 3.38−
3.25 (AB system, 2H), 3.16−2.67 (m, 2H), 2.56 (app tt, J = 8.1, 8.1,
(+)-Ethyl 2-[(Benzyloxycarbonyl)(1-tert-butoxycarbonyl-(S)-
piperidin-3-yl)amino]acetate ((S)-4). Et3N (290 mg, 2.9 mmol)
in dichloromethane (2 mL) was added to a solution of (S)-1 (745 mg,
2556
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
2.6 mmol) in dichloromethane (8 mL) at 0 °C, and N-
(benzyloxycarbonyloxy)succinimide (648 mg, 2.6 mmol) was added
in portions. The reaction mixture was stirred at room temperature for
17 h, diluted with dichloromethane (30 mL), and washed with brine
(2 × 15 mL). The organic phase was dried (Na2SO4), filtered, and
concentrated in vacuo. Purification by flash chromatography (heptane/
ethyl acetate 2:1, Rf = 0.23) gave (S)-4 as a transparent oil (0.94 g,
84%). Analytical chiral HPLC (hexane/2-propanol 95:5) tR 18.7 min,
98% purity, ee 100%. 1H NMR (rotamers) δ 7.48−7.19 (m, 5H), 5.18
and 5.11 (rot, s, 2H), 4.31−3.79 (m, 6H), 2.80−2.41 (m, 3H), 2.04−
1.89 (m, 1H), 1.80−1.32 (m, 12H), 1.27 and 1.17 (rot, t, J = 7.1 Hz,
3H). 13C NMR δ 169.8, 155.9, 155.1, 154.5, and 154.4 (rot), 136.2,
128.3, and 128.2 (rot), 127.8, 127.6, 79.5, 67.5, and 67.1 (rot), 61.0,
53.2, and 53.0 (rot), 47.1 and 46.5 (rot), 45.6 and 45.4 (rot), 43.3,
(−)-2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-leucin-
yl)-(R)-piperidin-3-yl)amino]acetamide ((R)-6). Compound (R)-6
was synthesized from (R)-5 (200 mg, 0.38 mmol) and NH3 in
methanol (7 N) (4.0 mL) as described for (S)-2. Purification by flash
chromatography (dichloromethane/methanol 95:5, Rf = 0.09) gave
(R)-6 as a white solid (122 mg, 65%). Analytical chiral HPLC
1
(hexane/ethanol 60:40) tR 12.3 min, 95% purity, de 90%. H NMR
(CD3OD, 50 °C) δ 7.39−7.23 (m, 5H), 5.12 (br s, 2H), 4.49−4.44
(m, 1H), 4.11−3.80 (m, 3H), 3.18−3.04 (m, 1H), 3.03−2.89 (m, 1H),
2.77−2.61 (m, 1H), 2.56−2.40 (m, 1H), 2.03−1.91 (m, 1H), 1.85−
1.27 (m, 15H), 1.02−0.73 (m, 6H). 13C NMR (CD3OD, 50 °C) δ
174.4, 173.7, and 173.4 (rot), 157.6, 137.8, 129.5, 129.1, 128.8, 80.5,
68.7, 54.9, 50.2, 47.6, 46.7, and 46.4 (rot), 43.1, 42.4, 28.8, 26.6, 25.9,
25.5, 23.5, 22.2; [α]D20 −33.9 (c 1.0, CH3OH). IR (KBr) v 3414, 2956,
1707, 1640, 1446 cm−1. HRMS (FT-ICR-MS) Calcd for C26H40N4O6
[M + H]+ 505.3021; found 505.3021.
20
28.7, 28.2, 24.4, 13.9; [α]D +14.0 (c 1.0, CHCl3). IR (neat) v 3499,
2976, 2863, 1747, 1693, 1415, 1152 cm−1; HRMS (FT-ICR-MS) calcd
for C22H32N2O6 [M + H]+ 421.2333; found 421.2334.
(+)-2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-leucin-
yl)-(S)-piperidin-3-yl)amino]acetamide ((S)-6). Compound (S)-6
was synthesized from (S)-5 (156 mg, 0.29 mmol) and NH3 in
methanol (7 N) (4.0 mL) as described for (S)-2. Purification by flash
chromatography (dichloromethane/methanol 95:5, Rf = 0.07) gave the
product as a white solid (108 mg, 73%). Analytical chiral HPLC
(−)-Ethyl 2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-
leucinyl)-(R)-piperidin-3-yl)amino]acetate ((R)-5). Deprotection
of (R)-4 (870 mg, 2.07 mmol) with TFA (2.0 mL, 27.0 mmol) in
dichloromethane (8 mL) was performed as described for (S)-5 to give
the Boc-deprotected intermediate. The crude intermediate and Boc-L-
Leu × H2O (551 mg, 2.21 mmol) were coupled as described for (S)-5
using EDC (424 mg, 2.21 mmol), HOBt (299 mg, 2.21 mmol), and
Et3N (813 mg, 8.04 mmol) in dichloromethane (11 mL) with
molecular sieves (600 mg). Purification by flash chromatography
(heptane/ethyl acetate 1:1, Rf = 0.2) gave (R)-5 as a transparent oil
(1.0 g, 91% yield over two steps). Analytical chiral HPLC (hexane/
ethanol 70:30) tR 7.5 min, 99% purity, de 97%. 1H NMR (CD3OD) δ
7.41−7.25 (m, 5H), 5.19−5.10 (m, 1H), 5.06 (s, 2H), 4.64−4.38 (m,
2H), 4.23−3.84 (m, 6H), 3.22−3.04 (m, 1H), 3.03−2.91 (m, 1H),
2.76−2.61 (m, 1H), 2.55−2.41 (m, 1H), 1.99−1.56 (m, 2H), 1.55−
1.07 (m, 14H), 1.00−0.72 (m, 6H). 13C NMR (CD3OD) δ 173.7 and
173.4 (rot), 171.8 and 171.7 (rot), 157.6, 157.2, 137.8, 129.5, 129.1,
128.8, 80.5, 68.6, 62.3, 55.0, 50.2, 46.7, 46.0, 43.1, 42.4, and 42.1 (rot),
1
(hexane/ethanol 60:40) tR 8.5 min, 100% purity. H NMR (CD3OD,
50 °C) δ 7.46−7.23 (m, 5H), 5.12 (br s, 2H), 4.49−4.38 (m, 1H),
4.20−3.79 (m, 3H), 3.26−3.10 (m, 1H), 3.09−2.93 (m, 1H), 2.77−
2.62 (m, 1H), 2.56−2.38 (m, 1H), 2.05−1.61 (m, 4H), 1.60−1.24
(12H), 1.00−0.75 (m, 6H). 13C NMR (CD3OD, 50 °C) δ 174.4,
173.8, and 173.5 (rot), 157.7, 137.8, 129.5, 129.0, 128.8, 80.4, 68.7,
54.7, 50.6, 47.4, 46.4, 43.6, 42.8, and 42.2 (rot), 28.8, 26.3, 26.0, 25.6,
20
23.6, 22.1; [α]D +13.2 (c 1.0, CH3OH). IR (KBr) v 3421, 2957,
1703, 1638, 1456 cm−1. HRMS (FT-ICR-MS) calcd for C26H40N4O6
[M + H]+ 505.3021; found 505.3022.
(−)-2-[(1-L-Leucinyl-(R)-piperidin-3-yl)amino]acetamide ×
2TFA ((R)-LpipG) ((R)-7). Compound (R)-6 (115 mg, 0.23 mmol)
was deprotected as described for (R)-LLpipG ((R)-10) to give the
white TFA salt of (R)-LpipG ((R)-7) (95 mg, 84%). Analytical
reversed phase HPLC (the mobile phase was 6.4−32.8% CH3CN in
water during 30 min, 0.085% TFA throughout) tR 6.2 min, 100%
20
29.0, 28.7, 26.0, 25.4, 23.6, 22.2, 14.4; [α]D −31.5 (c 1.0, CH3OH).
IR (KBr) v 3423, 2959, 1753, 1708, 1642, 1445, 1205 cm−1. HRMS
(FT-ICR-MS) calcd for C28H43N3O7 [M + H]+ 534.3174; found
534.3176.
1
purity. H (CD3OD) NMR δ 4.75 (app d, 1H), 4.48−4.38 (m, 1H),
3.98−3.86 (m, 2H), 3.74 (app d, 1H), 3.23−3.08 (m, 2H), 2.85 (app t,
1H), 2.32−2.18 (m, 1H), 1.98−1.51 (m, 6H), 1.05−0.95 (m, 6H). 13C
NMR (CD3OD) δ 169.9, 168.5, 54.7, 50.5, 46.6, 46.4, 44.5, 41.0, 28.0,
25.3, 25.0, 23.5, 21.6; [α]D20 −11.2 (c 1.0, CH3OH). IR (KBr) v 3420,
2967, 1676, 1202, 1134 cm−1. HRMS (FT-ICR-MS) calcd for
C13H26N4O2 [M + H]+ 271.2129; found 271.2128. For NMR spectral
assignment, see the Supporting Information.
(+)-Ethyl 2-[(Benzyloxycarbonyl)(1-(tert-butoxycarbonyl-L-
leucinyl)-(S)-piperidin-3-yl)amino]acetate ((S)-5). TFA (2.0 mL,
27.0 mmol) was added to (S)-4 (950 mg, 2.26 mmol) in
dichloromethane (8 mL) and stirred at room temperature for 16 h.
The solvent and excess of reagents were removed in vacuo to give the
Boc-deprotected intermediate. Boc-L-Leu × H2O (620 mg, 2.49
mmol), EDC (477 mg, 2.49 mmol), and HOBt (336 mg, 2.49 mmol)
were stirred in dichloromethane (6 mL) with molecular sieves (600
mg) at 0 °C for 15 min. Into this solution, the Boc-deprotected
intermediate and Et3N (9.14 mg, 9.04 mmol) in dichloromethane (5
mL) were added and the reaction mixture was stirred at room
temperature for 15 h. The mixture was diluted with dichloromethane
(20 mL) and washed with aqueous citric acid (10%) (2 × 10 mL),
saturated aqueous NaHCO3 (2 × 10 mL), and brine (1 × 10 mL). The
organic phase was dried (Na2SO4) and filtered. The solvent was
removed in vacuo. Purification by flash chromatography (heptane/
ethyl acetate 1:1, Rf = 0.18) gave (S)-5 as a transparent oil (950 mg,
79% yield over two steps). Analytical chiral HPLC (hexane/ethanol
70:30) tR 6.4 min, purity 100%. 1H NMR (CD3OD) δ 7.49−7.22 (m,
5H), 5.44−5.15 (m, 1H), 5.07 (s, 2H), 4.72−4.47 (m, 2H), 4.24−3.65
(m, 6H), 3.58−3.30 (m, 1H), 3.12−2.81 (m, 1H), 2.69−2.48 (m, 1H),
2.47−2.23 (m, 1H), 2.12−1.90 (m, 2H), 1.89−1.10 (m, 15H), 1.06−
0.72 (m, 6H). 13C NMR (CD3OD) δ (174.0, 173.8 and 173.4, rot),
171.8, 171.6, (157.7, 157.0 and 156.8, rot), 137.8 and 137.6 (rot),
129.5 and 129.4 (rot), 129.2 and 129.1 (rot), 128.8 and 128.7 (rot),
80.4 and 80.1 (rot), 68.5 and 68.3 (rot), 62.2, 54.5, and 54.3 (rot),
50.4, 48.0, 46.4, and 46.2 (rot), 45.8, 30.1, 29.7, and 29.5 (rot), 28.8,
(+)-2-[(1-L-Leucinyl-(S)-piperidin-3-yl)amino]acetamide ×
2TFA ((S)-LpipG) ((S)-7). Compound (S)-6 (117 mg, 0.23 mmol)
was deprotected as described for (R)-LLpipG ((R)-10) but with
stirring for 14 h for the first deprotection. The second deprotection
gave the white TFA salt of (S)-LpipG ((S)-7) (87 mg, 75%).
Analytical reversed phase HPLC (the mobile phase was 6.4−32.8%
CH3CN in H2O for 30 min, 0.085% TFA throughout) tR 6.6 min,
1
100% purity. H (CD3OD) NMR δ 4.55−4.40 (m, 1H), 4.25 (app d,
1H), 4.00−3.85 (m, 2H), 3.66−3.57 (m, 1H), 3.50−3.42 (m, 1H),
2.63 (app t, 1H), 2.32−2.16 (m, 1H), 2.03−1.93 (m, 1H), 1.90−1.48
(m, 6H), 1.07−0.94 (m, 6H). 13C NMR (CD3OD) δ 170.3, 168.5,
20
54.9, 50.5, 46.7, 46,6, 43.9, 41.1, 27.3, 25.3, 23.5, 23.1, 21,7; [α]D
+14.8 (c 1.0, CH3OH). IR (KBr) v 3418, 2965, 1676, 1201, 1134
cm−1. HRMS (FT-ICR-MS) calcd for C13H26N4O2 [M + H]+
271.2129; found 271.2128.
(−)-Ethyl 2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-
leucinyl)-L-leucinyl)-(R)-piperidin-3-yl)amino]acetate ((R)-8).
Deprotection of (R)-5 (649 mg, 1.22 mmol) by TFA (1.0 mL, 13
mmol) in dichloromethane (6 mL) were performed as described for
(S)-5 to give the Boc-deprotected intermediate. The intermediate and
the second Boc-L-Leu × H2O (334 mg, 1.34 mmol) were coupled as
described for (S)-5 with EDC (257 mg, 1.34 mmol), HOBt (181 mg,
1.34 mmol), Et3N (493 mg, 4.87 mmol), and molecular sieves (200
mg) in dichloromethane (11 mL). Purification by flash chromatog-
20
26.2, 25.9, and 25.7 (rot), 23.7, 22.0, 14.5; [α]D +25.7 (c 1.0,
CH3OH). IR (KBr) v 3425, 2958, 1754, 1720, 1640, 1442, 1204 cm−1.
HRMS (FT-ICR-MS) calcd for C28H43N3O7 [M + H]+ 534.3174;
found 534.3174.
2557
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
raphy (heptane/ethyl acetate 1:1, Rf = 0.14) gave (R)-8 as a
68.6, 56.4, 54.7, 47.3, 46.4, 46.1, 43.5, 42.8, 42.1, 42.0, 29.1, 28.8, 26.2,
20
transparent oil (690 g, 88% yield over two steps). Analytical chiral
25.8, 25.7, 23.6, 23.4, 22.2, 22.0; [α]D −9.5 (c 0.67, CH3OH). IR
1
(KBr) v 3407, 2957, 1707, 1638, 1450 cm−1. HRMS (FT-ICR-MS)
calcd for C32H51N5O7 [M + H]+ 618.3861; found 618.3861.
HPLC (hexane/ethanol 70:30) tR 9.2 min, 100% purity. H NMR
(CD3OD) δ 7.43−7.25 (m, 5H), 5.20−5.02 (m, 2H), 5.00−4.85 (m,
1H), 4.65−4.51 (m, 1H), 4.50−4.38 (m, 1H), 4.21−3.80 (m, 8H),
3.21−3.03 (m, 1H), 3.02−2.89 (m, 1H), 2.79−2.61 (m, 1H), 2.52−
2.38 (m, 1H), 1.98−1.35 (m, 13H), 1.34−1.18 (m, 2H), 1.16−1.09
(m, 3H) 1.04−0.81 (m, 12H). 13C NMR (CD3OD) δ 174.9, 172.3,
and 172.1 (rot), 171.6 and 171.4 (rot), 157.5, 157.2, 157.0, and 156.7
(rot), 137.6, 129.4, 129.0, 128.8, 128.7, 80.2, 68.4, and 68.3 (rot), 62.2
and 62.1 (rot), 54.2, 54.5, 49.6, 46.5, 45.9, and 45.6 (rot), 42.8, 42.2,
and 41.9 (rot), 28.7, 26.4, and 25.7 (rot), 25.6, 25.4, 23.7, 23.6, 23.5,
22.2, 22.0, 21.4, 14.5; [α]D20 −43.5 (c 1.0, CH3OH). IR (KBr) v 3318,
2957, 1754, 1709, 1640, 1445, 1206 cm−1. HRMS (FT-ICR-MS) calcd
for C34H54N4O8 [M + H]+ 647.4014; found 647.4015.
(−)-2-[(L-Leucinyl-(1-L-leucinyl-(R)-piperidin-3-yl))amino]-
acetamide × 2TFA ((R)-LLpipG) ((R)-10). Compound (R)-9 (282
mg, 0.46 mmol) was deprotected as described for (S)-LpipGP ((S)-
18) but with stirring for 3 h during the first deprotection step.
Purification by flash chromatography (ethyl acetate/methanol 20:3)
gave the Cbz-deprotected intermediate (189 mg). Further depro-
tection (180 mg) was performed as described for (S)-LpipGP ((S)-
18) with TFA/dichloromethane (1:10) (3 mL) and with stirring for
14 h to give the white TFA salt of (R)-LLpipG ((R)-10) (251 mg,
90%). Analytical reversed phase HPLC (the mobile phase was 10.8−
37.2% CH3CN in H2O for 30 min with 0.1% TFA throughout) tR 8.3
1
(−)-Ethyl 2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-
leucinyl)-L-leucinyl)-(S)-piperidin-3-yl)amino]acetate ((S)-8).
Deprotection of (S)-5 (535 mg, 1.0 mmol) with TFA (1.0 mL, 13
mmol) and dichloromethane (6 mL) were performed as described for
the synthesis of (S)-5 to give the Boc-deprotected intermediate. The
intermediate and the second Boc-L-Leu × H2O (275 mg, 1.10 mmol)
were coupled as described for the synthesis of (S)-5 with EDC (212
mg, 1.10 mmol), HOBt (149 mg, 1.10 mmol), Et3N (406 mg, 4.02
mmol), and molecular sieves (240 mg) in dichloromethane (11 mL).
Purification by flash chromatography (heptane/ethyl acetate 1:1, Rf =
0.24) gave (S)-8 as a transparent oil (590 g, 91% yield over two steps).
Analytical chiral HPLC (hexane/ethanol 70:30) tR 5.6 min, 100%
purity. 1H NMR (CD3OD) δ 7.47−7.23 (m, 5H), 5.28−5.02 (m, 2H),
5.00−4.86 (m, 1H), 4.67−4.50 (m, 1H), 4.48−4.36 (m, 1H), 4.23−
3.97 (m, 6H), 3.96−3.81 (m, 1H), 3.74−3.62 (m, 1H), 3.49−3.36 (m,
1H), 3.27−3.17 (m, 1H), 3.05−2.90 (m, 1H), 2.73−2.60 (m, 1H),
2.54−2.35 (m, 1H), 2.14−1.08 (m, 18H), 1.00−0.74 (m, 12H). 13C
NMR (CD3OD) δ 174.8, 172.6, and 172.5 (rot), 171.7 and 171.5
(rot), 157.5, 156.8, and 156.5 (rot), 137.6 and 137.5 (rot), 129.3,
129.1, and 128.9 (rot), 128.7, 128.6, 80.2, 68.5, 68.3, and 68.1 (rot),
62.1, 54.3, 50.2, 46.3, 46.2, 45.7, 43.4, 42.7, 41.9, 30.0, 28.8, 26.2, 25.8,
min, 100% purity. H NMR (CD3OD) δ 4.93−4.82 (m, 1H), 4.67
(app d, 1H), 4.09 (app d, 1H), 4.04−3.83 (m, 3H), 3.82−3.64 (m,
2H), 3.47−3.38 (m, 1H), 3.26−3.09 (m, 2H), 2.84 (app t, 1H), 2.32−
2.11 (m, 1H), 1.99−1.41 (m, 6H), 1.04−0.76 (m, 12H). 13C NMR
(CD3OD) δ 172.8, 170.7, 168.6, 55.6, 54.8, 52.7, 46.7, 46.4, 44.5, 41.8,
20
41.3, 28.0, 25.7, 25.1, 25.0, 23.5, 23.1, 21.9, 21.7; [α]D −13.6 (c 1.0,
CH3OH). IR (KBr) v 3422, 2964, 1676, 1202, 1135 cm−1. HRMS
(FT-ICR-MS) calcd for C19H37N5O3 [M + H]+ 384.2969; found
384.2967. For NMR spectral assignment, see the Supporting
Information.
(+)-2-[(1-(L-Leucinyl-L-leucinyl)-(S)-piperidin-3-yl)amino]-
acetamide × 2TFA ((S)-LLpipG) ((S)-10). Compound (S)-9 (230
mg, 0.37 mmol) was deprotected as described for (R)-LLpipG ((R)-
10) to give the white TFA salt of (S)-LLpipG ((S)-10) (209 mg,
92%). Analytical reversed phase HPLC (the mobile phase was 10.8−
37.2% CH3CN in H2O for 30 min) tR 8.60 min, 100% purity. 1H NMR
(CD3OD) δ 4.83−4.77 (m, 1H), 4.53−4.39 (m, 1H), 4.13−3.86 (m,
3H), 3.75−3.65 (m, 1H), 3.63−3.45 (m, 2H), 3.39−3.32 (m, 1H) 2.60
(app t, 1H), 2.31−2.14 (m, 1H), 2.09−1.97 (m, 1H), 1.94−1.81 (m,
1H), 1.80−1.56 (m, 5H), 1.55−1.39 (m, 1H), 1.04−0.86 (m, 12H).
13C NMR (CD3OD) δ 173.0, 171.3, 168.7, 56.1, 55.2, 52.7, 47.1, 46.9,
20
23.8, 23.6, 22.4, 22.2, 22.0, 14.5; [α]D −9.2 (c 0.5, CH3OH). IR
20
44.1, 41.6, 41.3, 27.5, 25.8, 25.2, 24.4, 23.5, 23.2, 22.1, 21.8; [α]D
(KBr) v 3319, 2957, 1753, 1709, 1641, 1447, 1205 cm−1. HRMS (FT-
ICR-MS) calcd for C34H54N4O8 [M + H]+ 647.4014; found 647.4014.
(−)-2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-leucin-
yl)-L-leucinyl)-(R)-piperidin-3-yl)amino]acetamide ((R)-9). Com-
pound (R)-9 was synthesized from (R)-8 (213 mg, 0.33 mmol) and
NH3 in methanol (7 N) (2.0 mL) as described for (S)-2, but the
reaction mixture was stirred for 168 h. Purification by flash
chromatography (dichloromethane/methanol 95:5, Rf = 0.05) gave
the product as a white solid (119 mg, 63%). Analytical chiral HPLC
(hexane/ethanol 70:30) tR 13.7 min, 100% purity. 1H NMR (CD3OD)
δ 7.42−7.24 (m, 5H), 5.20−5.05 (m, 2H), 4.96−4.87 (m, 1H), 4.66−
4.52 (m, 1H), 4.48−4.37 (m, 1H), 4.14−3.84 (m, 6H), 3.17−2.92 (m,
2H), 2.73−2.59 (m, 1H), 2.54−2.40 (m, 1H), 2.03−1.91 (m, 1H),
1.87−1.34 (m, 14H), 1.00−0.84 (m, 12H). 13C NMR (CD3OD, 50
°C) δ 175.0, 174.4, 172.6, and 172.4 (rot), 157.7, 157.5, 137.8, 129.5,
129.0, 128.7, 80.6, 68.7, 55.2, 54.8, 54.6, 47.6, 46.7, 46.1, 43.0, 42.3,
+14.3 (c 1.0, CH3OH). IR (KBr) v 3419, 2966, 1674, 1202, 1137
cm−1. HRMS (FT-ICR-MS) calcd for C19H37N5O3 [M + H]+
384.2969; found 384.2966.
Biological Studies. Purification of IdeS. For recombinant
IdeS expression an ideS allele was PCR-amplified from genomic
streptococcal DNA using primers 5′-TGTTACCATGGA-
TAGTTTTTCTGCTAAT-3′ introducing a NcoI restriction
site and 5′-GCTAGGTACCTTAATTGGTCTGATTCCAAC-
TAT-3′ introducing an Acc65I restriction site for subsequent
cloning into plasmid pETtrx_1a.29 IdeS was expressed in
Escherichia coli strain BL21 to obtain a His-tagged Trx-IdeS
protein. The fusion protein was purified on Ni2+-resin using
standard protocols. The His-Trx tag was removed by enzymatic
cleavage with Tev-protease in His-trap binding buffer (20 mM
Tris-HCl pH8.0, 150 mM NaCl, 10% glycerol) at 30 °C for up
to 20 h followed by a second round of purification on Ni2+-resin
to remove uncleaved fusion protein.29 The collected flow
through, containing IdeS without tag, was purified by size
exclusion chromatography on a HiPrep 16/60 Sephacryl S-300
HR column (GE Healthcare). Purified protein was kept in PBS
at −20 °C until use.
IdeS Activity Assay and Inhibitor Screening. IdeS (10 μL) (1.83
μM in PBS) was mixed with peptide or peptide analogue (3 μL) (30
mM in 50% DMSO, 50 mM phosphate buffer pH 7.4) and incubated
for 15 min at room temperature prior to the addition of IgG1 (10 μL)
(Sigma I5154, 8.07 μM in 20 mM TRIS-buffered saline pH 8.0) as
substrate. The reactions were incubated for 20 min at 37 °C before
termination by the addition of iodoacetamide (10 μL, 16.5 mM)
(Sigma I1149). Samples were run in triplicates. Controls where IdeS
had been replaced with PBS were included in duplicates for each
peptide/peptide analogue. Samples were analyzed by SDS-PAGE and
surface plasmon resonance spectroscopy.
20
42.1, 29.1, 28.7, 26.6, 26.0, 25.7, 25.3, 23.6, 23.3, 22.2, 21.9; [α]D
−50.0 (c 0.67, CH3OH). IR (KBr) v 3408, 2956, 1704, 1638, 1449
cm−1. HRMS (FT-ICR-MS) calcd for C32H51N5O7 [M + H]+
618.3861; found 618.3863.
(−)-2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-leucin-
yl)-L-leucinyl)-(S)-piperidin-3-yl)amino]acetamide ((S)-9). Com-
pound (S)-9 was synthesized from (S)-8 (394 mg, 0.61 mmol) and
NH3 in methanol (7 N) (4.0 mL) as described for (R)-7. Purification
by flash chromatography (dichloromethane/methanol 95:5, Rf = 0.13)
gave (S)-7 as a white solid (325 mg, 86%). Analytical chiral HPLC
1
(hexane/ethanol 70:30) tR 8.4 min, 100% purity. H NMR (CD3OD)
δ 7.51−7.22 (m, 5H), 5.27−5.06 (m, 2H), 4.98−4.86 (m, 1H), 4.66−
4.50 (m, 1H), 4.47−4.37 (m, 1H), 4.23−3.79 (m, 6H), 3.28−3.11 (m,
1H), 3.06−2.91 (m, 1H), 2.72−2.58 (m, 1H), 2.54−2.38 (m, 1H),
2.04−1.81 (m, 2H), 1.80−1.25 (m, 13H), 0.99−0.75 (m, 12H). 13C
NMR (CD3OD, 50 °C) δ 174.9, 174.3, and 174.2 (rot), 172.7 and
172.3 (rot), 157.5, 137.8, and 137.7 (rot), 129.4, 128.9, 128.7, 80.5,
2558
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
Determination of Inhibition Constants. Ki values were determined
by accessing velocity curves at different substrate concentrations in the
presence or absence of inhibitor. Inhibitor concentrations were 5, 2,
and 0.5 mM for (R)-LpipG ((R)-7) and 5, 2.5, and 1 mM for (S)-
pipG ((S)-3). For IdeS, repurified polyclonal IgG at concentrations of
150, 100, 50, 20, or 10 μM, was used in reactions with 2 μM of IdeS.
Reactions were analyzed by non- reducing SDS page gel analysis to
allow measurements of the initial substrate cleavage. The enzyme
velocity v was determined according to the following equation:
recorded. The final concentration of SpeB during the experiment was
0.28 μM The amount of active SpeB was determined by active-site
titration using various amount of the cysteine protease inhibitor E-64
as previously described.31 For inhibition assays, 11 μL of activated
SpeB (1.12 μM in PBS) was mixed with 2 μL of peptide or peptide
analogue (30 mM in 50% DMSO, 50 mM phosphate buffer pH 7.4)
and incubated for 15 min at room temperature prior to the addition of
substrate solution. All assays were performed in triplicate.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and characterization of compounds
shown in Schemes 3−6; NMR spectral assignments of (R)-1,
For papain, Nα-benzoyl-DL-arginine 4-nitroanilide hydrochlor-
ide (Sigma) at concentrations of 6, 3, 1, 0.5, or 0.25 mM was
used with 5 μM of papain and inhibitor. Ki nonlinear regression
(curve fit) for competitive inhibition and enzymatic kinetics
were calculated using GraphPad Prism version 5.04 for
Windows, GraphPad Software, San Diego California, www.
graphpad.com.
Surface Plasmon Resonance Spectroscopy Assays. Surface
plasmon resonance spectroscopy assays were performed with 1042.5
RU of Protein A (Abcam) covalently bound to a CM5 chip using
NHS/EDC coupling according to the manufacturer’s instructions. One
part of the IgG1/IdeS reaction was mixed with two parts water before
applying the sample on the Protein A-CM5 chip. Run conditions on
the Biacore X100 machine were 5 μL/min flow rate, a 90 s sample
injection time, HBS-EP+ (Biacore) as running buffer, and 20 mM
glycine pH 2.0 as regeneration solution of the chip. Report point for
amount of protein bound to the chip surface was retrieved at 190 s
after sample injection stop. Amount of IgG1 cleavage was first plotted
as percentage decrease between PBS-control (uncleaved IgG1) and
IdeS/IgG1 samples. A standard curve for IdeS concentration was made
without the presence of peptide or peptide analogue. The experimental
data obtained from assays with IdeS together with peptide/peptide
analogue experiments were plotted to the standard curve and
presented as approximate fraction of active IdeS at any given time
point.
Papain Activity Assay. Inhibition of the enzymatic activity of
papain by peptides and peptide analogues was determined by a
colorimetric assay. Papain (Fluka 76218) at a concentration of 1.23
μM was diluted in assay buffer (81 μL) (123 mM phosphate buffer pH
6.2, 1.23 mM EDTA, 1.23 mM DTT), mixed with peptides or peptide
analogues (16 μL) (30 mM in 50% DMSO, 50 mM phosphate buffer
pH 6.2) and incubated for 10 min at room temperature. The final
concentration of papain during the experiment was 0.9 μM. The
chromogenic papain substrate Nα-benzoyl-DL-arginine-4-nitroanilide
hydrochloride (3 μL) (Sigma B4875, 100 mM in DMSO) was added,
and the reaction was incubated for 2 h at 37 °C before termination by
the addition of glacial acetic acid (10 μL). Abs405 was measured to
determine the amount of cleaved substrate. Samples were run in
duplicates on two separate occasions.
Purification of SpeB. For purification of SpeB, S. pyogenes strain
5448 was grown for 16 h in Todd−Hewitt broth (TH) (BD
Biosciences) at 37 °C; 5% (v/v) CO2. The growth media was cleared
by centrifugation and subjected to ammonium sulfate precipitation
(50−80% (w/v)). The resulting pellet was dissolved in 1× PBS,
dialyzed o/n at 4 °C against 20 mM sodium acetate buffer, pH 5. The
sample was further purified on a HiTrap SP FF anion exchange
column (GE Healthcare), equilibrated in 20 mM sodium acetate, pH
5. Proteins were eluted by a linear NaCl gradient and SpeB eluted at
approximately 0.2 M NaCl. The eluted protein fraction was dialyzed
o/n against 1× PBS and kept at −20 °C.
1
(R)-2, (R)-3, (R)-4, (R)-7, and (R)-10; H and 13C NMR
spectra of tested compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +46-31-786 9031. Fax: +46-31-772 1394. E-mail:
luthman@chem.gu.se.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Swedish Research Council
(project numbers 2005-3145 (K.L.) and 2009-4997 (UvPR)
and Insamlingsstiftelsen at Umea University. We thank
̊
AstraZeneca R&D Molndal, Sweden, for financial support of
̈
the graduate student fellowship for KB. We also thank
Madeleine Åhman, AstraZeneca, Sweden, for help with the
pKa measurements.
ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl; Cbz-OSu,
N-(benzyloxycarbonyloxy)succinimide; DMSO, dimethylsulf-
oxide; E64, 1-[N-[(3-trans-carboxyoxirane-2-carbonyl)-L-
leucyl]amino]-4-guanidinobutane; EDC, N-(3-methylamino-
propyl)-N′-ethylcarbodiimide; ee, enantiomeric excess; HOBt,
hydroxybenzotriazole; HPLC, high pressure liquid chromatog-
raphy; IdeS, immunoglobulin G-degrading enzyme of Strepto-
coccus pyogenes; NMR, nuclear magnetic resonance; pip, the
piperidine moiety replacing glycine residues; rac, racemic; SDS-
PAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresis; SpeB, streptococcal pyogenic exotoxin B; TFA,
trifluoroacetic acid; TLCK, tosyl lysyl chloromethyl ketone;
TPCK, tosyl phenylalanyl chloromethyl ketone; Z-LVG CHN2,
N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethylketone
REFERENCES
■
(1) Rawlings, N. D.; Tolle, D. P.; Barrett, A. J. MEROPS: the
peptidase database. Nucleic Acids Res. 2004, 32, D160−D164.
(2) Cunningham, M. W. Pathogenesis of group A streptococcal
infections. Clin. Microbiol. Rev. 2000, 13, 470−511.
(3) Carapetis, J. R.; Steer, A. C.; Mulholland, E. K.; Weber, M. The
global burden of group A streptococcal diseases. Lancet Infect. Dis.
2005, 5, 685−694.
(4) Telfer, N. R.; Chalmers, R. J. G.; Whale, K.; Colman, G. The role
of streptococcal infection in the initiation of guttate psoriasis. Arch.
Dermatol. 1992, 128, 39−42.
(5) Elliott, S. D. A proteolytic enzyme produced by group A
streptococci with special reference to its effect on the type-specific M
antigen. J. Exp. Med. 1945, 81, 573−592.
SpeB Activity Assay. SpeB activity was measured essentially as
previously described.30 Briefly, SpeB was activated with 2 mM
dithiothreitol (DTT) for 30 min at 37 °C and mixed with 150 μL of 1
mM substrate solution, n-benzoyl-proline-phenylalanine-arginine-p-
nitroanilide hydrochloride (BPFA) (pH 4) (Sigma), in 0.1 M
phosphate buffer (pH 6). The change in absorbance at OD405 was
2559
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560

Journal of Medicinal Chemistry
Article
(6) von Pawel-Rammingen, U.; Johansson, B. P.; Bjorck, L. IdeS, a
novel streptococcal cysteine proteinase with unique specificity for
(26) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a
useful metric for lead selection. Drug Discovery Today 2004, 9, 430−
431.
̈
immunoglobulin G. EMBO J. 2002, 21, 1607−1615.
(27) Wan, H.; Holmen
́
, A. G.; Wang, Y. D.; Lindberg, W.; Englund,
(7) Wenig, K.; Chatwell, L.; von Pawel-Rammingen, U.; Bjorck, L.;
̈
M.; Nagard, M. B.; Thompson, R. A. High-throughput screening of
̊
̊
Huber, R.; Sondermann, P. Structure of the streptococcal
endopeptidase IdeS, a cysteine proteinase with strict specificity for
IgG. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17371−17376.
(8) Agniswamy, J.; Nagiec, M. J.; Liu, M. Y.; Schuck, P.; Musser, J.
M.; Sun, P. D. Crystal structure of group A Streptococcus Mac-1: insight
into dimer-mediated specificity for recognition of human IgG.
Structure 2006, 14, 225−235.
pKa values of pharmaceuticals by pressure-assisted capillary electro-
phoresis and mass spectrometry. Rapid Commun. Mass Spectrom. 2003,
17, 2639−2648.
(28) Meloun, M.; Bordovska, S. Benchmarking and validating
algorithms that estimate pK(a) values of drugs based on their
molecular structures. Analyt. Bioanalyt. Chem. 2007, 389, 1267−1281.
(29) Bogomolovas, J.; Simon, B.; Sattler, M.; Stier, G. Screening of
fusion partners for high yield expression and purification of bioactive
viscotoxins. Protein Expression Purif. 2009, 64, 16−23.
(9) Kagawa, T. F.; Cooney, J. C.; Baker, H. M.; McSweeney, S.; Liu,
M. Y.; Gubba, S.; Musser, J. M.; Baker, E. N. Crystal structure of the
zymogen form of the group A Streptococcus virulence factor SpeB: An
integrin-binding cysteine protease. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 2235−2240.
(30) North, M. J. Cysteine endopeptidases of parasitic protozoa.
Methods Enzymol. 1994, 523−539.
(31) Nyberg, P.; Rasmussen, M.; von Pawel-Rammingen, U.; Bjorck,
̈
(10) Olsen, J. G.; Dagil, R.; Niclasen, L. M.; Sørensen, O. E.;
Kragelund, B. B. Structure of the mature streptococcal cysteine
protease exotoxin mSpeB in its active dimeric form. J. Mol. Biol. 2009,
393, 693−703.
L. SpeB modulates fibronectin-dependent internalization of Strepto-
coccus pyogenes by efficient proteolysis of cell-wall-anchored protein F1.
Microbiology 2004, 150, 1559−1569.
́
(11) Collin, M.; Olsen, A. Effect of SpeB and EndoS from
Streptococcus pyogenes on human immunoglobulins. Infect. Immun.
2001, 69, 7187−7189.
(12) Vincents, B.; von Pawel-Rammingen, U.; Bjorck, L.;
̈
Abrahamson, M. Enzymatic characterization of the streptococcal
endopeptidase, IdeS, reveals that it is a cysteine protease with strict
specificity for IgG cleavage due to exosite binding. Biochemistry 2004,
43, 15540−15549.
(13) Burton, D. R. Immunoglobulin-Gfunctional sites. Mol.
Immunol. 1985, 22, 161−206.
(14) Rasmussen, M.; Bjorck, L. Proteolysis and its regulation at the
̈
surface of Streptococcus pyogenes. Mol. Microbiol. 2002, 43, 537−544.
(15) Agniswamy, J.; Lei, B. F.; Musser, J. M.; Sun, P. D. Insight of
host immune evasion mediated by two variants of group A
Streptococcus Mac protein. J. Biol. Chem. 2004, 279, 52789−52796.
(16) Potempa, J.; Dubin, A.; Korzus, G.; Travis, J. Degradation of
elastin by a cysteine proteinase from Staphylococcus aureus. J. Biol.
Chem. 1988, 263, 2664−2667.
(17) Johansson, B. P.; Shannon, O.; Bjorck, L. Ides: A bacterial
̈
proteolytic enzyme with therapeutic potential. PLoS ONE 2008, 3,
e1692.
(18) Nandakumar, K. S.; Johansson, B. P.; Bjorck, L.; Holmdahl, R.
̈
Blocking of experimental arthritis by cleavage of IgG antibodies in
vivo. Arthritis Rheum. 2007, 56, 3253−3260.
(19) Yang, R.; Otten, M. A.; Hellmark, T.; Collin, M.; Bjorck, L.;
̈
Zhao, M.-H.; Daha, M. R.; Segelmark, M. Successful treatment of
experimental glomerulonephritis with IdeS and EndoS, IgG-degrading
streptococcal enzymes. Nephrol., Dial., Transplant. 2010, 25, 2479−
2486.
(20) Berggren, K.; Johansson, B.; Fex, T.; Kihlberg, J.; Bjorck, L.;
̈
Luthman, K. Synthesis and biological evaluation of reversible inhibitors
of IdeS, a bacterial cysteine protease and virulence determinant. Bioorg.
Med. Chem. 2009, 17, 3463−3470.
(21) Filira, F.; Biondi, L.; Gobbo, M.; Rocchi, R. N-Alkylation of
amino acids during hydrogenolytic deprotection. Tetrahedron Lett.
1991, 32, 7463−7464.
(22) Hamid, M.; Slatford, P. A.; Williams, J. M. J. Borrowing
hydrogen in the activation of alcohols. Adv. Synth. Catalysis 2007, 349,
1555−1575.
(23) Berggren, K.; et al., unpublished results.
(24) The peptides were purchased from CASLO Laboratory ApS,
Lyngby, Denmark.
(25) Åkesson, P.; Moritz, L.; Truedsson, M.; Christensson, B.; von
Pawel-Rammingen, U. IdeS, a highly specific immunoglobulin G
(IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by
specific IgG antibodies generated during infection. Infect. Immun.
2006, 74, 497−503.
2560
dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560